Accurate differentiation of fusion transcripts derived from chromosome translocations is of critical importance for optimizing treatment strategies for leukemia. However, as the number of leukemia associated translocations is steadily increasing, single-tube, multiplex detection of multiple fusion gene transcripts becomes challenging. The overall goal of this proposal is to develop an assay system for rapid risk-stratification of chromosomal translocations in leukemia. The system combines multiplex RT-PCR with multiplex signal detection on fluorescent beads in a 96-well plate format. Clinical decision-making is based on the relative expression level of a specific fusion target to the internal or exogenous controls. The assay offers significant improvements in speed and cost over currently available assays and can be applied for a high-throughput risk-stratification in leukemia diagnostics. This assay is flexible to be easily adapted to other nucleic-based assays useful for cancer/diseases diagnostics. Specific aims for Phase I: (1) Develop a rapid single-tube assay for simultaneous identification of common chromosome translocations related to leukemia in a single tube (up to 21 translocations). (2) Develop a comprehensive control system for the clinical test of this assay. In Phase II we will adapt this assay into a robust, high-throughput clinic diagnostic kit for rapid risk stratification in leukemia. Our second aim for Phase II is to produce armored RNAs for exogenous control and all positive target controls using well-established in-house system and test the amplification and detection efficiency of these armored RNA controls. The use of armored RNA ensures the stability of our positive controls in the assay system.